PCR-based method of detecting murine calicivirus

ABSTRACT

The invention disclosed herein relates to a newly discovered murine  norovirus,  and compositions and methods related thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/757,832, entitled, “Murine Calicivirus” filed Jan. 14, 2004, now U.S.Pat. No. 7,041,444, which claims the benefit of priority to ProvisionalU.S. Patent Application Ser. No. 60/440,016, filed Jan. 14, 2003, whichapplications are hereby incorporated herein by reference in theirrespective entireties.

REFERENCE TO GOVERNMENT GRANT

This invention was made with government support under Grant No. RO1A149286. The United States government may have certain rights in theinvention.

BACKGROUND

The Caliciviridae are a family of positive-sense, single-stranded RNAviruses with a 7-8 kb genome that are divided into 4 distinct genera andfurther subdivided into genogroups. The genera Norwalk-like viruses,together with the closely related Sapporo-like viruses, recently renamedNoroviruses and Sapoviruses (Mayo, M. A., Arch. Virol. 147:1655-1656,2002), make up human caliciviruses (Kapikian, A. Z. et al., J. Virol.10:1075-1081, 1972; Jiang, X. et al., Science 250:1580-1583, 1990;Jiang, X. et al., Virol. 195:51-61, 1993; Hardy, M. E. et al., VirusGenes 12:287-290, 1996). Noroviruses are responsible for more than 90%of all cases of non-bacterial epidemic gastroenteritis(Kapikian et al.,1972; Kapikian, A. Z. et al., Chapter 25 in Fields Virology, Fields, B.N. et al., Eds., 1996; Pang, X. L. et al., Pediatr. Infect. Dis. J18:420-426, 1999; Pang, X. L. et al., J. Infect Dis. 181(Supp.2):S288-S294, 2000; Fankhauser, R. L. et al., J. Infect. Dis.178:1571-1578, 1998; Glass, R. I. et al., J. Infect. Dis. 181(Supp.2):S254-S261, 2000; Hedlund, K. O. et al., J. Infect. Dis. 181(Supp.2):S275-S280, 2000; Koopmans, M. et al., J. Infect. Dis. 181(Supp.2):S262-S269, 2000; Inouye, S. et al., J. Infect. Dis. 181(Supp.2):S270-S274, 2000). There are no current therapeutic drugs or vaccinesfor these important human pathogens. Sapoviruses are typicallyassociated with sporadic cases of pediatric gastroenteritis (Pang etal., 1999; Pang et al., 2000). Two other calicivirus genera, Vesivirusesand Lagoviruses, contain animal viruses exclusively. Calicivirus genomestypically contain a large 5′ open reading frame (ORF1) encoding anonstructural polyprotein, followed by ORF2 encoding a single capsidprotein. ORF2 is either in frame with ORF1 or present as an independentORF. While the 5′ end of ORF1 shows extensive sequence diversity, theremainder of ORF1 contains motifs arranged in a specific order conservedbetween caliciviruses and picornaviruses. ORF3, encoding a basicprotein, is present at the 3′ end of the genome preceding a poly-A tract(Clarke, I. N. et al., J. Infect. Dis. 181(Supp. 2):S309-S316, 2000).

DESCRIPTION OF THE FIGURES

FIG. 1: Passage of a new pathogen by intracranial inoculation inRAG/STAT−/− and IFNαβγR−/− mice.

The unknown pathogen was passaged into RAG/STAT−/− and IFNαβγR−/− miceand caused lethal disease within 30 days of inoculation (A),characterized histologically by meningitis (C), vasculitis of thecerebral vessel (D), and encephalitis (E) compared to mock-infectedbrain (B). (B, C) RAG/STAT−/− mice; (D, E) IFNαβγR−/− mice. Brainhomogenate from an infected RAG/STAT−/− mouse was passed into 129wild-type mice (A) and sera of these mice harvested 35 days later testednegative for mycoplasma, Sendai virus, reovirus type 3, Theiler's mouseencephalomyelitis virus (GDVII strain), lymphocytic choriomeningitisvirus, pneumonia virus of mice, minute virus of mice, mouse hepatitisvirus, ectromelia virus, epizootic diarrhea of infant mice, mousecytomegalovirus, polyoma virus, K virus, orphan parvovirus, and mouseadenovirus.

FIG. 2: Sequencing and phylogenetic analysis of the MNV-1 genome.

A) Double-stranded cDNA (dsDNA) from the brain of an infected IFNαβγR−/−mouse at passage 2 (FIG. 1) was prepared, digested with restrictionenzymes, and ligated to adaptors containing PCR primer sequences togenerate “tester” nucleic acids. dsDNA lacking linkers was preparedconcurrently from a control brain to generate “driver” nucleic acids.Serial rounds of subtractive hybridization of tester in the presence ofexcess driver followed by PCR amplification of tester-specific sequenceswere performed to generate difference products (DP) one through four(DP1-DP4). DP3 and DP4 were cloned into pGEMT (Promega, Madison, Wis.),sequenced, analyzed using BLAST, and clones (1-8, FIG. 2A) homologous,but not identical, to calicivirus sequences were identified that spannedthe Norwalk virus genome. Sequences within RDA clones (indicated byasterisks) were used to clone and sequence five fragments (a′, b′, c′,d′, e′, FIG. 2A) of the MNV-1 genome after PCR or 5′ and 3′ RACE(Marathon cDNA amplification kit, Clontech, Palo Alto, Calif.). The 5′end of the genome was difficult to clone and consequently the first 15nucleotides are based on a single sequence, while the remaining sequencehas at least a 10-fold redundancy. This may explain why there is nostart codon close to the 5′ end as is expected based on comparison withother Noroviruses. B) Schematic of the final 7726 bp MNV-1 genomesequence with predicted open reading frames (ORFs). The locations ofamino acid motifs in ORF1 are indicated: 2C helicase: GXXGXGKT (SEQ IDNO: 50); 3C protease: GDCG (SEQ ID NO: 51); 3D polymerase: KDEL (SEQ IDNO: 52), GLPS (SEQ ID NO: 53), YGDD (SEQ ID NO: 54). The putative S andP domains of the ORF2 encoded capsid protein were identified based onsequence alignments with Norwalk virus. AAA: 3′ poly-A tail. C)Alignment of the complete MNV-1 genome with complete genomes ofrepresentative members of the four Caliciviridae genera and members ofthe most closely related virus family, the Picornaviridae. Specificmembers were chosen based on the 2000 taxonomy study by Green et al. (JInfect Dis '00 v. 81 p. S322). D) Alignment of the capsid proteinsequence of MNV-1, done as in C. Note that the alignments in C and Dwere confirmed using other algorithms (data not presented).

FIG. 3: Sequence variability of MNV-1.

A) All variable nucleotides within ORF1 and ORF2, based on sequenceanalysis of multiple clones of the entire MNV-1 genome, are depicted.These nucleotides had 20% or less variability between clones. B)Sequences of individual clones spanning nucleotides 1767 to 1893 (solidbox on ORF1 in panel A), with variable positions highlighted witharrowheads SEQ ID Nos:21-48). The consensus sequence of MNV-1 is shownat the bottom (bold type) (SEQ ID NO:49), with variable nucleotideshighlighted by arrowheads.

FIG. 4: Purification and pathogenicity of MNV-1.

MNV-1 was purified from an infected IFNαβγR−/− mouse brain homogenate byCsCl density gradient centrifugation. As a control, mock-infected mousebrain homogenates were processed similarly. (A) Determination of theaverage buoyant density of genome-containing MNV-1 particles. Dialyzedgradient fractions were analyzed by MNV-1 specific RT-PCR (Titaniumone-step RT-PCR kit, Clontech, Palo Alto, Calif.) and products wereseparated on a 1% agarose gel. Primers were chosen in ORF1 to yield anexpected product of 184 bp (indicated by the asterisk). (B) MNV-1virions visualized by EM. Samples were absorbed ontoformvar/carbon-coated grids for 1 min. The grids were washed in dH₂O,stained with 2% aqueous uranyl acetate (Ted Pella Inc., Redding, Calif.)for 1 min, and air dried prior to viewing on a JEOL 1200EX transmissionelectron microscope (JEOL USA, Peabody, Mass.). (C) Survival ofRAG/STAT−/− mice infected i.c. with unpurified, or purified MNV-1, aswell as gradient fractions from mock-infected brain. The P values formock versus infected mice are indicated. Statistical analyses wereperformed using GraphPad Prism software.

FIG. 5: IFNαβ or IFNγ receptors and STAT1 are required to protect fromlethal MNV-1 challenge.

A MNV-1 stock was prepared as a brain homogenate from 17 IFNαβγR−/− miceinoculated i.c. three days previously with brain homogenate from apassage 2 (FIG. 1) mouse. Infected brains were homogenized in sterilePBS and filtered through a 0.2 μm filter. Brains from five IFNαβγR-−/−mice inoculated i.c. with uninfected brain tissue were used to generatea mock virus stock. Mice of various strains were inoculated with MNV-1or mock-inoculated using 10 μl intracerebrally (ic), 25 μl intranasally(in), or 25 μl perorally (po). A number of mouse strains did not showincreased mortality compared to wild-type 129 controls (A). The survivalafter inoculation with MNV-1 or mock virus is shown for IFNαβγR−/− mice(B), STAT1−/− mice (C), RAG/STAT−/− mice (D), and STAT1/PKR−/− mice (E).All p values for mock versus infected groups were ≦0.0001 except:IFNαβγR−/− i.n.: p=0.002; STAT1−/− i.n.: p=0.097; and STAT1−/− p.o.:p=0.034. Statistical analyses were performed with GraphPad Prismsoftware.

FIG. 6: Generation of MNV-1 virus-like particles.

A) Western blot analysis of cell lysates from High-Five cells infectedwith recombinant baculovirus expressing the MNV-1 capsid protein (seeExample 9) or a control baculovirus expressing the LacZ cassette(negative control). Proteins were detected by ECL Plus after incubationwith serum from a MNV-1 infected mouse followed by a HRP-labeledsecondary antibody. The size of the molecular weight marker is indicatedon the right. B)-D) Electron microscopy of negatively stained VLPs.Supernatants of High-Five cells infected with a control baculovirusexpressing LacZ (B), recombinant baculovirus expressing the MNV-1 capsidprotein (C), or VLPs purified from these supernatants (D) were stainedwith uranyl acetate and photographed at a magnification of 50,000×.

FIG. 7: Reactivity of mouse serum against MNV-1 VLP supernatants or celllysates by ELISA.

Supernatants of High-Five cells infected with recombinant baculovirusexpressing the MNV-1 capsid protein or LacZ expressing control werecoated on ELISA plates. A) Analysis of half-log serial dilutions ofserum from MNV-1 infected mice or 129 wild type mice. B) Analysis of1:10 dilution of several cages of STAT−/− mice. Each dot represents onemouse. Reactivity was assessed after incubation with a HRP-coupledsecondary antibody and calorimetric detection at 405 nm. Cages 1, 3, 4,5 and 6 contained seronegative mice. Cages 2, 7, 8, and 9 containedseropositive mice.

FIG. 8: Tissue MNV-1 RNA levels after infection via different routes.

Four IFNαβγR −/− mice were inoculated with MNV-1 i.c. (10 μl), p.o. (25μl), or i.n. (25 μl). Two mice were sacrificed at both 2 and 7 dpi andlung (Lu), intestine (Int), brain (Br) and feces were collected. RNA wasextracted from each organ, and cDNA was synthesized and used (5 ng) intriplicate real time PCR reactions. Primers specific to a 131 nucleotideregion of ORF1 were used (sense=cagtgccagccctcttat (SEQ ID NO:19);antisense=gtcccttgatgaggagga (SEQ ID NO:20)). Signal was compared to astandard curve generated using a plasmid containing target sequences.Triplicate reactions were performed using GAPDH primers to verifyequivalent amounts of starting template (not shown). The levels of virusRNA as log₁₀ MNV-1 genome copies are shown (open bars=2 dpi, solidbars=7 dpi, *-undetectable levels).

FIG. 9: Immunohistochemical staining of spleen sections from MNV-1infected mouse. Formalin-fixed spleen sections from a STAT1−/− animal 3days after p.o. inoculation with MNV-1 were stained with either immunepolyclonal rabbit serum inoculated with bacterially expressed MNV-1capsid protein (left panel), or with the preimmune serum from the samerabbit (right panel). Immunohistochemistry was performed with thePerkinElmer™ TSA™-Plus DNP (HRP) System, according to the suppliedprotocol. Primary antibodies were used at a 1:25 dilution. Positivecells are indicated by arrows.

FIG. 10: Single copy sensitivity of MNV-1 cDNA detection by nested PCRassay. Nested PCR primers specific to a region of MNV-1 ORF2 weredesigned (outer-sense=gcgcagcgccaaaagccaat (SEQ ID NO:15);outer-antisense=gagtcctrtggcatgctacccagg (SEQ ID NO:16);inner-sense=gccgccgggcaaattaacca (SEQ ID NO:17); andinner-antisense=ggcttaacccctaccttgccca (SEQ ID NO:18)). A) Multiple PCRreactions with either 1 or 10 copies of a plasmid containing theappropriate region of MNV-1 were performed. 3/4 and 4/4 reactions werepositive for 1 and 10 copies, respectively. The expected size of the PCRproduct is 153 bp. B) cDNA was generated from spleen tissue of 10IFNαβγR−/− mice and 1 μg of each was used in nested PCR reactions (7/10samples were positive). All water controls are negative.

DESCRIPTION

It has been discovered that mice doubly deficient in STAT1 and RAG2(RAG/STAT) contained an infectious pathogen that caused severeencephalitis and could be serially passaged by intracerebral (i.c.)inoculation (FIG. 1). Lethal infection was associated with encephalitis,vasculitis of the cerebral vessels, meningitis, hepatitis, and pneumonia(FIG. 1 and data not shown). Disease was passed by filtered samples,suggesting the presence of a virus (FIG. 1A). Sera of 129 mice infectedwith the putative virus tested negative for an extensive panel of mousepathogens (see legend of FIG. 1). Brain homogenate from an infectedRAG/STAT−/− mouse was passed into 129 wild-type or IFNαβγR−/− micebefore and after filtration. A full work-up was performed on mice frompassages 1 and 2, including histopathology, electron microscopy,standard clinical virology and microbiology work-ups, as well as specialstains of histology sections (GMS, AFB [acid-fast bacilli], Gram stain).All of these failed to reveal the nature of the pathogen.

The pathogen is more virulent in mice lacking both the interferon αβ(IFNαβ) and the interferon γ (IFNγ) receptors (IFNαβγR−/−, ²) than inwild-type mice (see below) and it passes through a 0.2 μn filter (seeabove and FIG. 1A). The pathogen does not appear to cause cytopathiceffect on HeLa cells, Vero cells or murine embryonic fibroblasts(including those lacking IFN receptors or STAT1). These data suggestthat a previously unknown IFN-sensitive but non-cultivatable pathogenthat was <0.21 μm in size was present in diseased mice.

Identification and Sequencing

To identify the new pathogen a previously published representationaldifference analysis protocol (RDA) was used (See Pastorian et al., Anal.Bicochem. 283:89-98 (2000), which is hereby incorporated in itsentirety). Double-stranded cDNA (dsDNA) from the brain of an infectedIFNαβγR−/− mouse at passage 2 (FIG. 1) was prepared, digested withrestriction enzymes, and ligated to adaptors containing PCR primersequences (tester) (see Pastorian protocol for sequences of RDAprimers). Control dsDNA lacking linkers was prepared concurrently from acontrol brain (driver). Serial rounds of subtractive hybridization oftester in the presence of excess driver followed by PCR amplification oftester-specific sequences were performed to generate difference products(DP) one through four (DP1-DP4). DP3 and DP4 were cloned and sequenced.Three of 24 clones from DP3 and ten of 48 clones derived from DP4 hadsignificant homology to multiple caliciviruses (data not shown). TheseRDA clones spanned the Norwalk virus genome (FIG. 2A), but were notidentical to any known full or partial calicivirus sequence,demonstrating that we had identified a novel calicivirus. This new virusis referred to herein as murine Norovirus-1 (MNV-1).

To determine the relationship of MNV-1 to other caliciviruses, the MNV-1genome was cloned and sequenced from cDNA of an infected mouse brainusing a combination of 5′ and 3′ RACE and PCR (FIG. 2A). Sequencing wasperformed in both directions with 10-fold redundancy to obtain aconsensus sequence with the exception that the 5′ 15 nucleotides wereobtained from a single clone. The assembled genome included 7726 bp ofunique sequence plus a 3′ polyA tail, and contained the expected threeORFs conserved across the Caliciviridae (FIG. 2B). Phylogenetic analysisusing the CLUSTAL W algorithm, and other algorithms including PAUP,aligning either the complete genome sequence (FIG. 2C) or the capsidprotein sequence (FIG. 2D) of MNV-1 with corresponding sequences frommembers of the four calicivirus genera and several members of thePicornaviridae family revealed that MNV-1 is a Norovirus that does notcluster within previously identified genogroups (FIG. 2C, D)(Green K YJID 181 S322-330). Therefore, it is proposed that MNV-1 is exemplary ofa new Norovirus genogroup.

Thus, disclosed herein is a pathogen that infects mice, referred toherein as Murine Norovirus-1 (MNV-1). MNV-1 is both a unique norovirus,and is the first member of a new genogroup of Noroviruses. An exemplarysequence for the MNV-1 virus and genogroup is provided as SEQ ID NO:1,which is a consensus sequence representative of the full length MNV-1genome as determined from a series of clones derived by PCR or RACEanalysis from RNA derived from the brain of an infected mouse. Thus, oneembodiment comprises an isolated RNA sequence as shown in SEQ ID NO:1.An additional embodiment comprises sequences of MNV-1 isolates that varyfrom the sequence in SEQ ID NO:1 by an amount determined by bothsequence analysis and current understanding of the relatedness ofdifferent caliciviruses (see below). One embodiment comprises theviruses related directly to MNV-1 as viral quasispecies. Anotherembodiment comprises other members of the MNV-1 genogroup of which MNV-1is the defining member. The criteria for viral quasispecies and viralgenogroup are defined below, and serve to specifically set criteria forthe MNV-1 embodiments described herein.

RNA viruses vary during infection due to errors made by the viralRNA-dependent RNA polymerase. Thus, MNV-1 (a positive-strand RNA virus)may be expected to vary during replication into a quasispeciescomprising multiple viruses with sequences closely related to, but notidentical to, the sequence of the original infecting virus. Thus, someembodiments of MNV-1 include viruses with sequences that vary from thesequence provided in SEQ ID NO:1 by an amount consistent with variationwithin a calicivirus quasispecies. The level of variation from the MNV-1consensus SEQ ID NO:1 that still is considered by those skilled in theart to be the same virus (since these viruses always exist asquasispecies) is 5-7% (Radford et Al. Veterinary record Jan. 29, 2000 pp117 on, Radford et al Vet Record Oct. 20, 2001 pp 477 on). Thus, anembodiment comprises the MNV-1 viral quasispecies of sequences that varyfrom our initial consensus sequence (SEQ ID NO:1) by no more than 5%. Ithas been confirmed that there is significant variance in MNV-1nucleotide sequence even within a single infected animal (FIG. 3). Toshow this, a portion of the primary data from which the 10-foldredundant consensus sequence SEQ ID NO:1 was derived is presented. Itwas found that over the highly conserved ORF2 region, there are multiplesites at which there is sequence variation (FIG. 3A). A portion of thesequence data is presented in FIG. 3B for a region within which sequencevariation was found. The frequency of variation at the sites shown inboxes is greater than that observed at multiple other sites (e.g. theremainder of the sequence in FIG. 3B), showing that these variationsrepresent true biological variation rather than PCR artifacts orsequencing errors. Thus, MNV-1 does exist as a quasispecies.

Further embodiments comprise viruses with an amount of variance from SEQID NO:1 that is consistent with variation within a genogroup, and lessthan the variation observed between genogroups. For caliciviruses,genogroup and genus definition has been developed and officially set bythe International Committee on the Taxonomy of viruses (ICTV) andresearch in the field has led to definitions of the amount of variationin sequence that is expected within a single genogroup as opposed tobetween viruses of different genogroups (K. Y. Green et al JID 2000S322-330). Because nucleotide sequences can vary without causingvariation in amino acid sequence, relatedness at the nucleotide level isa preferred method for distinguishing between genogroups or within aquasispecies (see above). Nucleotide identity within a genogroup ofNoroviruses has been established as greater than 80% within the highlyconserved capsid protein (ORF2) gene (J. Vinje et al Arch Virol (2000)145:223-241). Thus, viruses that differ by more than 20% at thenucleotide level from a member of a genogroup (in this case from theMNV-1 sequence in SEQ ID NO:1) are not members of the genogroup.Nucleotide identity between genogroups is 64%-78% or less. Therefore,the genogroup to which MNV-1 belongs comprises viruses that vary by nomore than 20% from the MNV-1 sequence within the capsid region. Similarreasoning applies to other conserved regions of the genome including theRNA dependent RNA polymerase gene. Therefore, our use of the capsidsequence for the definition of genogroup is standard.

Further embodiments include RNA sequences that are at least about 80%identical to SEQ ID NO:1, where the % identity is determined usingVector NTI AlignX program. Other embodiments include an isolated DNAsequence, or fragments thereof, identical to or complementary to SEQ IDNO:1, and isolated DNA sequences at least about 80% identical to orcomplementary to SEQ ID NO:1. Further embodiments comprise sequencesbetween 80% and 100% identical to SEQ ID NO:1, and sequencescomplementary thereto.

Additional embodiments comprise fragments of any of the above mentionedsequences, such as may be used, for example, as primers or probes.Examples of such sequences include the primers listed in legends toFIGS. 8 and 10 that were used to detect virus infection in animals bynested PCR (FIG. 10) or to determine the amount of MNV-1 genome in atissue by the use of real time PCR (FIG. 8). These primers will beuseful for detection of MNV-1 infection in commercially bred mice andfor quantitation of MNV-1 in tissues after trials of antiviral agents orvaccines. Such primers and probes are selected such that they aresubstantially complementary to a target sequence, wherein the targetsequence consists of coding sequence of MNV-1. Here, substantiallycomplementary means that the primer or probe is sufficientlycomplementary to the target sequence that it will hybridize to thetarget sequence under highly stringent conditions. As used herein,highly stringent conditions are as defined in the nested and real timePCR protocols exemplified in FIGS. 8 and 10. For hybridization in blotsas opposed to PCR reactions, stringent refers to: hybridization at 68degrees C. in 5×SSC/S× Denhardt's solution/1.0% SDS, and washing in0.2×SSC/1.0% SDS at room temperature. Such probes and primers areuseful, for example in various assays for detecting the presence ofMNV-1 (FIG. 10) and determining how much MNV-1 is in a particular sample(FIG. 8). Other assays for which such primers or portions of MNV-1sequence would be useful include Northern and Southern hybridizationblot assays, additional PCR assays (e.g. degenerate PCR using primerswith degenerate nucleotides at specific sites within the PCR primer todetect viruses within the MNV-1 genogroup but not identical to the MNV-1sequence in SEQ ID NO:1), transcription-mediated amplification assaysand the like, and as positive controls and internal standards forcommercial assays to detect the presence of MNV-1 in mice or aftertreatment with anti-viral agents or vaccines.

A feature that distinguishes the human Noroviruses from the Sapovirusesare the cup-shaped depressions on the virion surface that gave thecalicivirus family its name (calyx=cup in Latin). Sapovirus capsids showthese characteristic cup-shaped depressions by electron microscopy (EM),while Norovirus capsids have a feathery appearance. To visualize MNV-1virions, MNV-1 was purified from the brain of an infected IFNαβγR−/−mouse on CsCl gradients (FIG. 4). Gradient fractions containing MNV-1genome were identified by RT-PCR (FIG. 4A), revealing a buoyant densityof MNV-1 of 1.36 g/cm³ ×/− 0.04 g/cm³ (n=3 experiments), in closeagreement with the published buoyant densities of Noroviruses (1.33-1.41g/cm³). Analysis of these gradient fractions by EM revealed particleswith a diameter of 28-35 nm (FIG. 4B), similar to the known size (26-32nm) of Norovirus particles in negatively stained preparations. Theparticles were icosahedral and had the same feathery surface morphologyas Noroviruses but lacked the cup-like depressions characteristic ofSapoviruses. Gradient fractions prepared from mock-infected brain didnot contain these particles (data not shown).

To test the pathogenicity of MNV-1, mice were infected i.c. with CsClgradient purified MNV-1. These virions were infectious since 18/18RAG/STAT mice inoculated with them died, while 18 of 18 mice inoculatedwith gradient fractions prepared from a mock-infected brain survived(FIG. 4C). Mice inoculated with gradient-purified virions showedencephalitis, meningitis, cerebral vasculitis, pneumonia, and hepatitis(data not shown). This mortality rate and pathology was similar to thatobserved previously in mice inoculated with unpurified brain homogenate(FIG. 4C and data not shown). The presence of disease in mice inoculatedwith CsCl-purified MNV-1 demonstrates that MNV-1 is the causative agentof the disease initially detected and passed (FIG. 1).

The MNV-1 genome comprises three open reading frames (ORFs). Analysis ofthe predicted coding sequence of ORF1 indicated a polyprotein with amolecular weight (MW) of 180.7 kDa and revealed the presence of multipleconserved motifs shared by caliciviruses and picornaviruses (FIG. 2B).ORF2 is separated from ORF1 by 32 nt and starts in the −1 frame relativeto ORF1. It encodes a 542 aa protein with a calculated MW of 58.9 kDaand an isoelectric point of 5.19. When this gene was expressed in arecombinant baculovirus, virus-like particles (VLPs) were found in thesupernatant of infected cells, demonstrating ORF2 encodes the capsidprotein (FIG. 6). These VLPs provide a reagent for analysis of MNV-1infection (see below). The predicted ORF3 encodes a basic protein (pI of10.22) with a calculated MW of 22.1 kDa that overlaps by 2 nt with ORF2and is expressed in the +1 frame relative to ORF1 but the −2 framerelative to ORF2.

Thus, further embodiments comprise the amino acid sequences encoded byORF1, ORF2 and ORF3. These amino acid sequences are shown in SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4, respectfully. Additional embodimentscomprise amino acid sequences that are encoded by viruses that vary fromSEQ ID NO:1 by no more than 20% at the nucleotide level as definedabove. The protein translation of such sequences will vary on apercentage basis depending on the placement of nucleotides within codonsand the frequency of amino acids coded for by single versus multiplethree base pair codons used by the translational machinery. Thereforethe extent of variation of these embodiments is properly determined bydefining the extent of total nucleotide variation accepted as definingthe MNV-1 genogroup. Some embodiments comprise the nucleotide sequencesthat encode each of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3and SEQ ID NO:4, including degenerate variants that encode those aminoacid sequences. Additional embodiments comprise the nucleotide sequencesof ORF1, ORF2 and ORF3 of MNV-1.

Additional embodiments include vectors capable of expression of any ofthe proteins encoded by MNV-1 or their variants as defined above.Examples of suitable vectors include baculovirus vectors, alphavirusvectors (e.g. Sindbis virus vectors, VEEV replicons), retroviralvectors, plasmids within which expression is driven from eukaryoticpromoters, plasmids that generate short RNA sequences suitable for geneinactivation by RNAi technology, plasmids in which the presence of anRNA polymerase transcribes MNV-1 sequences (including the entiresequence) in order to provide RNA (including up to full lengthinfectious RNA) for analysis or transfection into cells. Infectious RNAis defined as RNA, which, on transfection into eukaryotic cells, givesrise to intact infectious virus. Portions of the genome may also beexpressed in this fashion for the generation of viral proteins or foranalysis of the processing of MNV-1 viral proteins for the purpose ofdeveloping assays for identification of steps in viral replication thatmay serve as drug targets. Additional uses of expression vectors includegeneration of cells expressing viral proteins in a stable fashion forthe purpose of screening anti-viral antibodies or for providing positivecontrols for assay for detection of anti-viral antibody in the serum.

As discussed above, expression of the capsid protein, i.e., the proteinencoded by the sequence of ORF2, results in the formation of virus-likeparticles (VLPs). Thus, some embodiments comprise methods of producingVLPs. Such methods comprise transfecting a cell or animal with a vectorthat encodes the MNV-1 capsid protein, and recovering VLPs, orexpression of the capsid protein from within the baculovirus genome suchthat the capsid protein is produced in insect cells infected with thebaculovirus expressing the capsid protein. Further embodiments compriseMNV-1 VLPs. VLPs are useful, for example, for isolation of antibodies,analysis of the epitopes that antibodies recognize, and for cryo-EM andX-ray crystallography and other methods for determining the threedimensional structure of the MNV-1 capsid. VLPs may also be studied forpotential use as a vaccine. In this setting they may be useful formapping the specific conformational epitopes recognized by anti-viralantibodies and the specific peptides recognized by antiviral CD4 and CD8T cells.

Antibodies

Some embodiments comprise antibodies that bind specifically to any ofthe various proteins encoded by the MNV-1 genome. Methods for producingantibodies are known in the art. Such antibodies may be eithermonoclonal or polyclonal. Antibodies can be used in various assays, suchas for example ELISA assays, to detect the presence of MNV-1 in asample. Samples include for example serum, saliva, feces, and tissues.In addition, antibodies may be utilized in methods for preventing lethalMNV-1 infection and studied for potential use as vaccines or anti-viraltherapeutics.

An example of the use of antibodies and antibody detection assays is thedemonstration that seroconversion can be detected by ELISA of serumusing MNV-1 VLPs as the target antigen bound to the ELISA plate (FIG.7). A further example is the demonstration that MNV-1 infection can bedetected in specific cells by using immunohistochemistry to detect thebinding of MNV-1 specific antibodies to infected cells (FIG. 9). Thistype of use may also be employed for detecting binding of virus to cellsby FACS analysis. This in turn will provide an opportunity to identifythe receptor for MNV-1. Identification of the MNV-1 receptor on the cellsurface may provide important targets for anti-viral drug development.In addition, antibodies will be used for immunofluorescence and in-situdetection of virus infected cells.

Small Animal Model

The discovery of the first murine Norovirus provides the first smallanimal model for development and testing of pharmaceuticals and vaccinesfor treatment and prevention of a major human disease. This presents anopportunity to answer important questions regarding the pathogenesis ofNorovirus infections, to determine the role and mechanisms of immunityin either protection against infection or immunopathology, to identifynovel therapeutic targets for treatment of human calicivirus disease,and to better understand how innate immunity can control enteric virusinfection. The mouse model can also be used in methods to identifyagents that alter calicivirus infection and disease.

The course of human Norovirus infection strongly suggests that symptomsare caused by acute infection. Prominent amongst the clinicalmanifestations are vomiting and diarrhea with a mean incubation periodof 24 hours and duration of 24-48 hours. Understanding of the viral andhost mechanisms involved in the induction and clearance of humanNorovirus disease is rudimentary. Acquired immunity can play a role inNorovirus resistance, but may not explain why certain individuals getsevere disease while others do not. It may be that long-term immunitycan be achieved, and the use of the MNV-1 virus in a small animal modelprovides the first opportunity to define such possible mechanisms.Infected individuals can develop short-term immunity to homologousvirus, but the development of long-term immunity is questionable. Anunexpected inverse relationship between pre-challenge antibody levelsand susceptibility to infection has been reported in some studies(Parrino, T. S., et al., N. Engl. J. Med. 297:86-89, 1977; Johnson, P.C. et al., J. Infect. Dis. 161:18-21, 1990; Okhuysen, P. C. et al., J.Infect. Dis. 171:566-569, 1995), while others have reported thatcirculating antibody does correlate with resistance to calicivirusinfection (Lew, J. F. et al., J. Infect. Dis. 169:1364-1367, 1994;Ryder, R. W. et al., J. Infect. Dis. 151:99-105, 1985;Nakata, S. et al.,J. Infect. Dis. 152:274-279, 1985). This controversy has led to studiesshowing that non-immune host factors, such as blood groups, influencesusceptibility to infection(Hutson, A. M. et al., J Infect. Dis.185:1335-1337, 2002). The discovery of MNV-1 provides a small animalmodel for the study of Noroviruses.

One embodiment is therefore the use of mice infected with MNV-1 as anapproach to identifying the efficacy of vaccines or therapeutic agents.Mice would be infected with the newly discovered virus, and then treatedwith candidate agents and the outcome of the infection monitored usingELISA (FIG. 7), quantitative real time PCR for the viral RNA (FIG. 8),immunohistochemistry (FIG. 9), lethality (FIG. 5), or in situhybridization to monitor the course of infection. Similarly, anotherembodiment is the use of mice infected with MNV-1 to test the efficacyof vaccination protocols against the virus. In this case, differentvaccine preparations (including vectors expressing portions of the newvirus genome or proteins from the virus or from human noroviruses thatcross react with proteins from the mouse virus) would be administered toinfected mice and the effect of vaccination on the course of theinfection monitored using ELISA (FIG. 7), quantitative real time PCR forthe viral RNA (FIG. 8), immunohistochemistry (FIG. 9), lethality (FIG.5), or in situ hybridization. As it is not practical to perform suchexperiments in humans, the capacity to perform these types of screensfor in vivo efficacy of therapeutics or vaccines is not possible withoutthe use of this newly described virus.

One embodiment includes nested PCR assays for determining presence,absence or quantity of MNV-1 in a tissue, organ or feces sample of amouse, wherein the organ is selected from the group consisting of lung,intestine and brain. These methods comprise subjecting a cDNAsynthesized from RNA comprised by the tissue, organ or feces of themouse to a first amplification reaction using a first sense primer and afirst anti-sense primer, wherein the first sense primer and the firstanti-sense primer hybridize to target sequences comprised by MNV-1. Thereaction product of the first amplification reaction can then besubjected to a second amplification reaction using a second sense primerand a second anti-sense primer, wherein the second sense primer and asecond anti-sense primer hybridize to target sequences comprised by areaction product of the first amplification reaction, if MNV-1 ispresent in the sample.

In addition, the discovery of MNV-1 and the generation of a consensussequence will enable construction of an infectious clone for MNV-1. Oneembodiment is therefore generation of such an infectious clone from thecurrent cloned and sequenced genome or from sequences that vary withinthe limits described above for the MNV-1 quasispecies or MNV-1genogroup. Such a clone can be used to develop various screening assaysfor MNV-1 antiviral agents and targets for antiviral drug developmentand vaccines for prevention of infection or antibodies for therapy ofdisease, and also may be used to study certain aspects of the virusesinfection cycle including binding, entry, uncoating, negative strand RNAsynthesis, positive strand RNA synthesis, subgenomic RNA synthesis,synthesis of structural and non-structural proteins, viral assembly andviral egress to be studied and used to develop screens for antiviraldrugs that might have uses in preventing or treating Norovirus induceddisease. In addition, placement of portions of human Noroviruses into aninfectious clone for MCV-1 (e.g. substituting proteins such as thecapsid of RNA polymerase of the human virus into the mouse virusinfectious clone) will allow the murine virus to be humanized andpotentially still used in mice. This will allow screening of therapeuticagents that target the functions of human norovirus proteins in ananimal model. This is possible only through the combined use of aninfectious MNV-1 clone as a vector for expressing functional proteinsand a small animal model which allows assessment of the effects oftherapeutic agents or vaccines on the course of infection with such“humanized” forms of the mouse calicivirus MNV-1.

In addition, the use of the newly discovered MCV-1 virus in mice withdifferent immune deficiencies will allow identification of host proteinsthat play a role in control of Norovirus infection. An example of thisis the detection of the critical role of STAT-1 in resistance to MNV-1infection (Working Example 14, FIG. 5). Identification of such hostproteins could allow development of targeted therapeutic agents thatenhance specific parts of the host immune response as a way to treat orprevent Norovirus disease. Such embodiments include, for example, use ofthe virus in mice with deficiencies in specific parts of the immunesystem in order to identify mice that have increased susceptibility orincreased resistance to infection by MCV-1. Such embodiments would beuseful for identifying immune protective or immunopathologic aspects ofthe host response and thereby inform searches for vaccines ortherapeutic agents that could prevent or treat Norovirus infection. Anexample would be targeting enhanced STAT-1 function, based on theexperiments in FIG. 5, for prevention of Norovirus disease in humans.

WORKING EXAMPLES Example 1 Generation of MNV-1 Stock

After identification of MNV-1 in RAG/STAT and IFNαβγR-deficient mice, abrain homogenate from an IFNαβγR-deficient mouse at passage 3 was usedfor i.c. inoculations of 17 additional IFNαβγR-deficient mice. Brains ofinfected mice were harvested 3 days post-infection and homogenized inPBS. Homogenates were centrifuged at low speed and filtered through a0.2 μm filter and the resulting supernatant was used in subsequentinfections. For control experiments, brain homogenates of mock-infectedmice were prepared similarly after injection of mice with uninfectedmouse brain homogenate. (See FIG. 5).

Example 2 Purification of MNV-1 Virions

Homogenate from one MNV-1 infected brain was used for purification ofMNV-1 virions while a mock-infected mouse brain was used as a control(FIG. 4). Homogenized brain was subjected to a cycle of freeze/thaw andtwo low speed centrifugations before filtration through 0.22 μm filter.Supernatants were centrifuged at 90,000×g for 2 hrs and the resultingpellets were incubated for 30 min at 37 C in 1 ml 1% Na-deoxycholate.The resulting material was mixed with CsCl to a final density of 1.36g/cm³ and centrifuged for 40 hrs at 150,000×g. Gradients werefractionated, their density determined with a refractometer, anddialyzed against a buffer containing 0.01M Tris-HCl, 0.15M NaCl, 1 mMCaCl₂, and 0.05M MgCl₂. (See FIG. 4).

Example 3 RNA Isolation, cDNA Synthesis, and RDA

Total RNA was isolated from a MNV-1 infected mouse brain using Trizol(Invitrogen, Carlsbad, Calif.) following the manufacturer'sinstructions. Double-stranded cDNA for use in RDA was synthesized fromtotal RNA using the Superscript Choice System for cDNA synthesis(Invitrogen, Carlsbad, Calif.) and a combination of random hexamers andoligo dT primers. Single-stranded cDNA for quantitative PCR wasgenerated using Supercript II (Invitrogen, Carlsbad, Calif.) followingthe manufacturer's recommendations. RDA was performed as described byPastorian et al. (Anal. Biochem. 283:89-98, 2000) with the followingmodification. The QIAquick PCR purification kit (Quiagen, Valencia,Calif.) was used to remove unincorporated nucleotides and small cDNAspecies. Difference products from rounds 3 and 4 were cloned into thepGEM-T vector system (Promega, Madison, Wis.) following themanufacturer's instructions. Bacterial colonies were grown up andinserts were PCR amplified for sequencing. (See FIG. 2, 3 ).

Example 4 RT-PCR and Quantitative PCR

RT-PCR was performed with primers 445/1/AS6(TCCAGGATGACATAGTCCAGGGGCG)(SEQ ID NO:5) and 445/1/S6(TGGGATGATTTCGGCATGGACAACG) (SEQ ID NO:6) using the Titanium one-stepRT-PCR kit (Clontech, Palo Alto, Calif.) following manufacturer'srecommendations. Quantitative PCR (FIG. 8) was performed with primersORF1/RT1/S (cagtgccagccctcttat) and ORF1/RT1/AS2 (tcctcctcatcaagggac)that amplify a 132 bp segment of ORF1 outside of the predictedsubgenomic RNA. This assay has a sensitivity of 100 viral genomes/μgcellular RNA or about 1 MNLV-1 genome per 1720 cell equivalents of RNA(estimating 1 μg cellular RNA/172,000 cells). The assay linearlyquantitates genome over at least a 6-log range. (See FIG. 8).

Example 5 Cloning of the MNV-1 Genome

A combination of PCR and RACE was used to clone the MNV-1 genome (FIG.2A). For internal sequence information, primers were constructed basedon sequence information obtained through RDA and used to amplify andclone larger pieces of MNV-1 from 1^(st) strand cDNA from a RAG/STATmouse brain (passage 3). These PCR products were cloned into the pGEMTvector (Promega, Madison, Wis.) and universal M13 forward and reverseprimers used for sequencing. Primer walking was applied when necessary.For the 5′ and 3′ ends of MNV-1, RACE was performed with the MarathoncDNA Amplification Kit (Clontech, Palo Alto, Calif.) using total RNAfrom the same RAG/STAT mouse brain (passage 3) as starting template.These products were cloned and sequenced as outlined above. A consensussequence with at least 10-fold redundancy (except for the 5′ end, seebelow) was constructed using the VectorNTI contig program. The 5′ end ofthe genome was difficult to clone and consequently the first 15nucleotides are based on a single sequence, possibly explaining whythere is no start codon close to the 5′ end as is expected based oncomparison with other Noroviruses. (See FIG. 2).

Example 6 Cloning and Expression of the MNV-1 Capsid Protein in Bacteria

The MNV-1 capsid protein was PCR amplified from first strand cDNA from aRAG/STAT mouse brain (passage 3). The following primers C-pET1(GTGGTGCTCGAGTGCGGCCGCAAGCTTTATTATTGTTTGAGCATTCGGCCTG) (SEQ ID NO:7) andN-pET1 (ATCCGAATTCTAGATGCACCACCACCACCACCACATGAGGATGAGTGATGGCGCA G) (SEQID NO:8) containing HindIII and EcoRI restriction sites (underlined),respectively, and a 6 Histidine N-terminal tag (bold) were used in a 2step PCR reaction (5 cycles 50 C, 30 cycles 60 C) in the presence of 5%DMSO. The resulting PCR product was ligated into a PCR blunt cloningvector (Zero Blunt PCR Cloning kit, Invitrogen, Carlsbad, Calif.) andtransformed into DH5α CaCl₂ competent cells (Invitrogen, Carlsbad,Calif.). DNA was isolated from the resulting clones and diagnosticrestriction digests followed by DNA sequencing confirmed the presenceand sequence of the insert. The insert was cloned into the bacterialexpression vector pET-30a (+) Novagen, Madison, Wis.) using the EcoRIand HindIII restriction sites. Next, BL21 (DE3) competent cells weretransformed and protein was expressed following the manufacturer'sprotocol (Novagen, Madison, Wis.).

Example 7 Purification of Bacterially Expressed MNV-1 Capsid Protein

Following a 2 hour expression, protein was purified from inclusionbodies of bacterial cells using the BugBuster protein extraction reagent(Novagen, Madison, Wis.). His-tagged capsid protein was isolated fromremaining protein by nickel column chromatography (Ni-NTA His BindResin, Novagen, Madison, Wis.) in the presence of 8M urea and proteaseinhibitors (protease inhibitor cocktail set III, Novagen, Madison,Wis.). Samples were dialyzed against 25 mM phosphate buffer (pH 6.0) andthe purity of each preparation was assessed by SDS-PAGE and silverstaining (Silver stain Plus kit, Biorad, Hercules, Calif.).

Example 8 Generation of Antisera in Rabbits

Rabbit sera was produced through Cocalico Biologicals, Inc. (Reamstown,Pa.). Basically, rabbits were injected with 100 μg bacterially expressedcapsid protein in CFA (complete Freund's adjuvant) and boosted after amonth once every month with 50 μg protein in IFA (incomplete Freund'sadjuvant). Production bleeds were collected a week after each boost andbefore the start of injections. The same procedure is being used forgeneration of antibodies directed against virus-like MNV-1 particles.

Example 9 Cloning and Expression of the MNV-1 Capsid Protein inBaculovirus

The MNV-1 capsid protein was cloned into the baculovirus expressionvector in an analogue way to the cloning into the bacterial expressionvector. The following primers were used for initial 2 step PCRamplification (4 cycles at 50 C, 30 cycles at 64 C) of the MNV-1 capsidprotein: N-Bac1 (CGGAATTCGGATGAGGATGAGTGATGGCGCA)(SEQ ID NO:9), C-Bac1(TCTCGACAAGCTTTTATTGTMTGAGCATTCGGCCT)(SEQ ID NO:10). The samerestriction sites, EcoRI and HindIII (underlined) were used for cloninginto the pFastBac1 vector (Invitrogen, Carlsbad, Calif.). Recombinantbaculoviruses were made using the Bac-to-Bac Expression system(Invitrogen, Carlsbad, Calif.) following the manufacturer'sinstructions. Briefly, the recombinant vector plasmid containing theMNV-1 capsid protein was transformed into DH10Bac E.coli cells allowingfor transposition of the gene of interest into the bacmid genome. Clonescontaining recombinant bacmids were identified by antibiotic selectionand disruption of the lacZ gene. Recombinant bacmid DNA was then usedfor transfection of Sf9 insect cells. Recombinant baculoviruses wereamplified for several rounds on Sf9 or Sf2 cells (Invitrogen, Carlsbad,Calif.) before infection of High-Five cells (Invitrogen, Carlsbad,Calif.) for protein expression. High-Five cells were infected for 5-7days and supernatant were collected for purification of MNV-1 VLPs.Initial preparations were screened for the presence of VLPs by negativestaining electron microscopy. VLPs were identified in the supernatantsof several isolates (FIG. 6C). Two isolates with the highest amount ofprotein expression were chosen for further experiments. The amount ofprotein expression in each preparation was analyzed by SDS-PAGE andimmunoblotting (FIG. 6A).

Example 10 Purification of MNV-1 VLPs

MNV-1 VLPs are purified from the supernatant of infected High-Five cells7 days post-infection (FIG. 6D). The purification protocol is based onLeite et al. (Arch Virol, 1996, 141:865-875), which is herebyincorporated by reference. Briefly, protein in the cell supernatant isbeing precipitated using PEG 8000, and particles are purified using CsClgradients. VLPs are dialyzed against 25 mM phosphate buffer, pH 6.0.Details of the protocol are being optimized at this point.

Example 11 Use of VLPs, Potential and Actual Targets of VLPs

VLP-containing insect cell supernatants are being used for ELISA toscreen mouse sera (see ELISA below). VLPs will be used to generaterabbit antisera. Their role as potential vaccine will be investigated.They will also be used for three-dimensional structure determination ofthe MNV-1 capsid.

Example 12 ELISA Assay

This assay can be used to screen mice capable of eliciting an antibodyresponse (FIG. 7). The assay was optimized for a maximalsignal-to-background ratio. VLP-containing insect cell supernatants areused as antigens for coating ELISA plates over night at 4 C. Plates areblocked for two hours at 37 C with 3% BSA and washed with 0.1 5MNaCl+0.05% Tween 20. Sera from mice are diluted 1:100 and incubated for1 hour at 37 C. After washing, wells are incubated for two hours at 37 Cwith a 1:1000 dilution of peroxidase-conjugated AffiniPure goatanti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., WestGrove, Pa.). Plates are developed after another round of washing byaddition of the substrate 2,2′-Azinobis 3-ethylbenzthiazoline sulfonicacid (ABTS, Sigma-Aldrich Corp., St. Louis, Mo.) for 10 min, thereaction is stopped using 0.2N phosphoric acid, and quantified byreading the absorbance at 415 nm.

Example 13 Nested PCR Assay

This assay can be used to screen tissues of immunocompromised mice withno antibody response (FIG. 10). RNA is isolated from the tissue(s) ofinterest and 1^(st) strand cDNA is being made (see above). To sets ofprimers were designed with the following sequences: outer sense primerCCAAAAGCCAATGGCTCTGA (SEQ ID NO:11), outer antisense primerAGTTGAATGGGCTCCAGGGT (SEQ ID NO:12), inner sense primerCCGCCGGGCAAATTAACCAA (SEQ ID NO:13), inner antisense primerAGGTGGGCAAGGTAGGGGTTA (SEQ ID NO:14). Each reaction contained 2 μl offirst strand cDNA and a final concentration of 1 μM sense and antisenseprimer, 2.5 mM MgCl₂, 0.2 mM dNTPs, and 1.25 unit Taq DNA Polymerase(Promega, Madison, Wis.) in 1× buffer (Taq DNA Polymerase 10× ReactionBuffer without MgCl₂, Promega, Madison, Wis.). PCR was performed for 45cycles for the 1^(st) round, and 30 cycles for the 2^(nd) round with thefollowing setting: heating 2 min 94 C, cycle for 30 sec 94 C, 30 sec 60C (annealing), and 30 sec 72 C (extension), with a final extension stepof 10 min 72 C. Two μl product from the 1^(st) round are used in the2^(nd) round using the same overall set-up. Products are analyzed byagarose gel electrophoresis.

Example 14 Use of MNV-1 Infected Mice as Small Animal Model of NorovirusInfection

To determine whether T and B cell mediated immunity is required forresistance to MNV-1, wild-type and RAG1−/− mice were infected by thei.c. route and followed for 90 days (data not shown). Surprisingly,MNV-1 infection does not kill RAG1−/− mice (n=20) after direct i.c.inoculation even though these mice are typically highly susceptible toinfection with a range of different viruses. The finding that RAG−/−mice are resistant to lethal disease argues that typical adaptiveresponses are not required for protection from lethal disease. Thisfinding may explain in part contradictory conclusions as to theimportance of antibody in resistance to Norovirus disease. While B and Tcell responses are not required for resistance to lethal infection, itmay be that pre-existing immunity influences the pathogenicity of MNV-1.Alternatively, the presence of immune cells may contribute to diseaseinduced by MNV-1 as is seen for lymphocytic choriomeningitis virus.

Together with a course of clinical illness too brief to allow typicaladaptive responses, these studies in RAG−/− mice beg the question ofwhether innate rather than acquired immunity is critical for resistanceto calicivirus infection. We therefore inoculated a variety of mousestrains lacking components of the innate immune system with MNV-1. Theperoral (p.o.) and intranasal (i.n.) routes were tested in addition tothe i.c. route since the physiologic routes of infection for humancaliciviruses are oral and respiratory. Mice lacking the IFNαβ receptoror the IFNγ receptor were no more susceptible to lethal infection thanwild-type controls (FIG. 5A). In contrast, mice lacking both IFNαβ andIFNγ receptors were more susceptible to lethal infection than congeniccontrols after either i.c. or i.n. inoculation with MNV-1 (FIG. 5B).These data demonstrate that the IFN receptors can compensate for eachother in resistance to MNV-1 infection such that only deficiency in bothreceptors leads to lethality. Mice deficient in inducible nitric oxidesynthetase (iNOS) or in the RNA activated protein kinase PKR, two IFNregulated proteins with antiviral properties, were also resistant tolethal MNV-1 infection after i.c. or p.o. inoculation (FIG. 5A).

Since deficiency in both IFN receptors is required to predispose tolethal MNV-1 infection, we reasoned that a component of the innateimmune system that can be activated by either the IFNαβ or the IFNγreceptor was critical for MNV-1 survival. We therefore tested thehypothesis that the latent cytoplasmic transcription factor STAT1, whichis shared by both the IFNαβ and IFNγ signaling pathways, was criticalfor resistance to calicivirus infection. STAT1 deficiency resulted inlethal MNV-1 infection in mice with T and B cells (STAT1−/−, FIG. 5C),mice lacking T and B cells (RAG/STAT, FIG. 5D), and mice lacking PKR(PKR−/−STAT−/−, FIG. 5E) by all routes analyzed. Thus STAT1 is the firsthost component identified as essential for resistance to lethalNorovirus infection, and is required for survival even when T and Bcells are present.

Having identified STAT1 as essential for calicivirus resistance, we theninvestigated the relationship between the interferon receptors andSTAT1. No statistically significant differences were found in thesurvival of IFNαβγR −/− and STAT1−/− mice after either i.c. or i.n.inoculations. However after p.o. inoculation, deficiency of STAT1, butnot deficiency in both IFNαβ and IFNγ receptors, led to lethal infection(see FIGS. 5B and C). This might suggest that STAT1 has IFNreceptor-independent effects that are critical for Norovirus resistance.Supporting this are findings that the biological effects of STAT1 do notcompletely overlap with those of the IFN receptors during viralinfection since there are both STAT1-independent antiviral effects ofthe IFN receptors, and IFN receptor-independent effects of STAT1.

1. A method for determining presence, absence or quantity of MNV-1 in atissue, organ or feces sample of a mouse, the method comprising: a)synthesizing cDNA from RNA comprised by the tissue, organ or fecessample; and b) detecting MNV-1 cDNA by a PCR assay if MNV-1 is presentin the sample, wherein the organ is selected from the group consistingof lung, intestine and brain.
 2. A method in accordance with claim 1,wherein the PCR assay is selected from the group consisting of a realtime PCR assay and a nested PCR assay.
 3. A method in accordance withclaim 1, wherein the PCR assay uses at least one sense primer and atleast one antisense primer, wherein the sequence of the sense primer isselected from the group consisting of SEQ ID NO:15, SEQ ID NO:17 and SEQID NO:19, and the sequence of the antisense primer is selected from thegroup consisting of SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20.
 4. Amethod in accordance with claim 3, wherein the sequence of the senseprimer is set forth as SEQ ID NO:15, and the sequence of the antisenseprimer is set forth as SEQ ID NO:16.
 5. A method in accordance withclaim 1, wherein the sequence of the sense primer is set forth as SEQ IDNO:17, and the sequence of the antisense primer is set forth as SEQ IDNO:18.
 6. A method in accordance with claim 1, wherein the sequence ofthe sense primer is set forth as SEQ ID NO:19, and the sequence of theantisense primer is set forth as SEQ ID NO:20.
 7. A method in accordancewith claim 2, wherein the PCR assay comprises a nested PCR assay,comprising: subjecting the cDNA to a first amplification reaction usinga first sense primer and a first anti-sense primer; and subjectingreaction product of the first amplification reaction to a secondamplification reaction using a second sense primer and a secondanti-sense primer, wherein the first sense primer and the firstanti-sense primer hybridize to target sequences comprised by MNV-1, andthe second sense primer and a second anti-sense primer hybridize totarget sequences comprised by a reaction product of the firstamplification reaction, if MNV-1 is present in the sample.
 8. A methodin accordance with claim 7, wherein the primers are specific to ORF2 ofan MNV-1.
 9. A method in accordance with claim 8, wherein the firstsense primer comprises the sequence set forth as SEQ ID NO: 15, thefirst anti-sense primer comprises the sequence set forth as SEQ ID NO:16, the second sense primer comprises the sequence set forth as SEQ IDNO: 17 and the second anti-sense primer comprises the sequence set forthas SEQ ID NO: 18.